An in vitro test, carried out by Biovet S.A. in collaboration with a private biotechnology laboratory in Spain, aimed to reveal the molecular mechanisms by which liver conditioner pronutrients promote a regenerative effect in liver cells that have been damaged experimentally with aflatoxin B1.

Aflatoxin B1 (AFB1) is a mycotoxin produced by the fungus Aspergillus flavus, and it is one of the most potent hepatotoxic and carcinogenic substances. AFB1 intercalates in DNA and induces mutations in the P56 gene, interfering in the activities of DNA polymerase and RNA polymerase and, thus, decreasing the mRNA and protein synthesis.

Pronutrients are molecules of plant origin that, through a metagenetic mechanism of action, cause the activation of a target cell, in this case the hepatocytes, and induce an increase in the translation of RNA to specific functional proteins.

To assess the effect of pronutrients on the mRNA-protein translation rate, a technique called SUnSET (Surface Sensing of Translation) was used.

The SUnSET technique is a non-radioactive method to monitor protein synthesis in cell populations, based on the use of puromycin. Puromycin is a tyrosyl-tRNA analog that can be incorporated into newly synthesized proteins through a peptide bond, so the detection of puromycin by an anti-puromycin antibody (a technique called ELISA Western Blot) can be correlated with the rate of translation mRNA-protein.

Image 1: Protein and protein synthesis with puromycin

PROCOTOL

• In vitro assays were performed in 24 wells, where 1.2 × 10⁵ cells/ml were added in a final volume of 2 ml per well.
• After 4 days of culture, confluence was observed and the medium was replaced with different supplements, following the scheme described below:
  • TREATMENT A (Negative control): No treatment.
  • TREATMENT B (Positive control): Only puromycin is added.
  • TREATMENT C (Challenge): Puromycin and aflatoxin B1 (AFB1) are added.
  • TREATMENT D (Challenge + pronutrients). Puromycin, aflatoxin B1, and liver conditioner pronutrients are added.

RESULTS AND INTERPRETATION

A darker signal in the ELISA Western Blot technique correlates with a higher amount of puromycin, and, therefore, a higher rate of RNA-protein translation.

• Treatment A does not show any protein synthesis as it does not contain puromycin that can serve as a marker.
• Treatment B shows basic protein synthesis thanks to the use of puromycin as a marker, without any supplement or challenge.
• Treatment C shows protein synthesis in aflatoxin B1 damaged hepatocytes.
• Treatment D shows an increase in protein synthesis (marked with a blue box) in hepatocytes, even though they have been damaged by aflatoxin B1, thanks to the presence of hepatic conditioner pronutrients.

CONCLUSIONS

In conclusion, liver conditioner pronutrients have shown an inductive effect on the mRNA-protein translation rate.

A higher rate of mRNA-protein translation correlates with an increase in the production of tissue-specific proteins. In this trial, where liver damage is induced, the increase shows that there is a greater regeneration process thanks to the use of these pronutrients.

This pronutrient-based liver conditioner, manufactured by Biovet S.A., is commercially available under the name Alquernat Livol.